It wasn’t that long ago when cellular miRNAs lived under the radar. They went to work on their targets each day and hung out with other members of the extended RISC family. Their small size and lack of poly-A tails may have kept them out of the spotlight for decades, but the last few years have been rough for miRNA.
Lately they’ve been labeled and manipulated more than a rising pop star. It all started out innocently enough, a ligation here, some sequencing there, but as the well deserved research infatuation has blossomed, these transcripts have been tagged, tailed and tweaked by researchers frothing to better understand the increasing roles miRNA play in cellular processes.
Today researchers definitely have options in their miRNA analysis toolkit, so let’s take a look at a few of the popular approaches, how these various methods differ, and how things are evolving in miRNA labeling and detection.
miRNA Microarray Prep: A Tail of Two Enzymes
T4 RNA Ligase: What do you do without a tail to prime? Adapt. Or should we say “adapter.” Using T4 RNA ligase, researchers first popped on adapters for Sanger sequencing and presto…miRNAs were being discovered left and right. The method worked well, was quick, and pretty easy, so it wasn’t long before researchers put T4 to work on the 3’ OH group of mature miRNAs. Using this approach, researchers were able to add a single fluoroescent dye molecule to the ends of miRNAs for microarray profiling (Nature Methods 2004). Today, several commercial solutions like Exiqon and Agilent’s microarray labeling kits use variations to this approach.
PolyA Polymerase (PAP): Another camp of researchers answered the lack of a poly-A substrate by adding one. By using Poly A polymerase (PAP), researchers were able to taaaaaaaail the ends of unsuspecting miRNAs.
The folks at Ambion borrowed a page out of previously used microarray protocols by incorporating amine-modified nucleotides during this tailing step that were later conjugated to the NHS ester forms of researchers favorite dyes (RNA 2005). The nice thing about the tailing approach was that multiple fluorescent molecules could be incorporated which should make for more sensitive profiling. That said, adding a huge tail on the end of a 21 bp miRNA can impact hybridization kinetics on microarrays, so you have to be careful about going overboard. Sure, you’ll have a ton of fluorescence on the end of the miRNAs, but they’ll migrate towards their respective probes like molasses if you’re not careful.
Branching Out with Fluorescent Dendrimers
Invitrogen scientists teamed up recently and released a protocol that takes bits from each of the earlier approaches in the NCode™ Rapid miRNA Labeling System. In this approach miRNAs are first tailed, but just enough to generate a substrate to which a fluorescent DNA “dendrimer” can be ligated. The dendrimer is a DNA polymer with 15 dye molecules spaced within it and with an oligo dT end so it can be tagged directly to the tailed miRNAs. The 15 dye molecules provide a nice signal boost, without all the drag of the longer tails of earlier methods and the process is about as quick as earlier direct tagging methods that added a single fluorophore to the miRNA targets.
Less Delivering More
What started out as a a technical hurdle in miRNA profiling (e.g. small size, absence of polyA tails) turned into a nice opportunity to tweak microarray labeling protocols for the better. We’re willing to bet if miRNAs didn’t require a different labeling approach they’d still be getting labeled the old fashioned way that requires hours more time and steps, leading to more variability in results.
More streamlined methods like the direct fluorescent tagging used in the NCode™ Rapid miRNA Labeling System have helped recent entrants to miRNA analysis, like Peter Nicholls down at Prince Henry’s Institute in Australia, to get a strong start to their work right out of the gates. “The NCode™ Rapid System has worked really well for us beginners as it was simple to use and removed potential for operator error. We’ve certainly got some nice results using this platform which we hope to publish very soon,” shared Nicholls.
For more details on miRNA Labeling products, check out the Product Database
Head to Invitrogen for more details on the NCode™ Rapid miRNA Labeling System